In Vitro and in Vivo Neutralization of Murine Activated Protein C

Abstract 3364

Activated Protein C (APC) is a circulating serine protease with two major roles to maintain homeostasis. APC acts via multiple receptors, including protease-activated receptor 1, to exert anti-apoptotic and vascular integrity protective effects. A number of protective effects of pharmacologic APC are reported in the literature, with beneficial effects in kidney, brain and irradiation-induced pathologies. The functional protections of the endogenous protein C systems are challenging to study. A better understanding of its mechanisms at different cellular levels and in different tissues is needed to enable evaluation of its further usage in humans. To that end, new tools should be considered to increase our knowledge. To help evaluate the endogenous murine protein C system and to be able to neutralize pharmacologic APC, we have made and characterized a novel rat monoclonal anti-mouse protein C antibody, SPC-54, that almost completely ablates in vitro and in vivo APC activity. In solid phase binding assays, the Kd of SPC-54 for APC was about 8 nM. In biochemical assays, SPC-54 inhibited amidolytic activity of wild-type murine APC by > 95%. SPC-54 was similarly a potent inhibitor (> 90%) of the amidolytic activity of the 5A-APC mutant. IC50 value for wild-type APC and the 5A-APC mutant were comparable. SPC-54 was pre-incubated with APC, followed by the addition of a 20 fold molar excess of biotinylated FPR-chloromethylketone, quantification of biotinylation of APC was readily made by SDS-PAGE and Western blots using infrared-coupled streptavidin. SPC-54 blocked successfully active site titration of APC using this biotinylated active site titrant. These and other experiments suggest that the SPC-54 epitope is located in the vicinity of the active site, such that it blocks different small substrates from reaching the active site. When we performed thrombin generation assays using mouse platelet-poor plasma to check whether SPC-54 was a potent blocker of APC activity in plasma, we showed that SPC-54 neutralized almost completely exogenous APC anticoagulant activity in a dose-dependent manner. Using native polyacrylamide gel migration, Western immunoblotting and immuno-precipitation with protein G-agarose, we confirmed that SPC-54 was bound to protein C in plasma after infusing mice with SPC-54 (10 mg/kg). Moreover, using a modified ELISA that is capable to quantify the pool of activatable protein C, the plasma protein C activity level was considerably decreased (> 80%) in mice after a single injection of SPC-54 (10 mg/kg), and that this effect of neutralizing circulating protein C was sustained for at least 7 days. For in vivo proof of concept, we performed murine tissue factor-induced thromboembolism experiments. Results showed a severe decrease in survival of mice that were pre-infused with SPC-54 when compared to control (survival time of 7 min vs. 42.5 min respectively, P = 0.0016). Moreover, blood perfusion in lungs of mice infused with SPC-54 (10 mg/kg) was dramatically impaired (decrease of 54%, P < 0.0001) as revealed by infrared quantification of Evans Blue dye as marker of vascular perfusion. We also used endotoxemia murine models to assess effects of SPC-54. SPC-54 decreased survival after endotoxin challenge (25 mg/kg, LD50 dose) in mice infused with SPC-54 (10 mg/kg) at 7 hours after LPS. Mortality was 100% after 36 h in the SPC-54 group, whereas controls, which received either boiled SPC-54 antibodies or PBS vehicle, showed a mortality of about 50% (P < 0.001). In summary, SPC-54 is a potent rat monoclonal antibody that neutralizes murine APC activities in vitro and in vivo. Its characteristic ability to dampen the endogenous protein C/APC system is of value to understand better the role of the endogenous protein C system in murine injury models and also to neutralize pharmacologic murine APC.